Feeling out a link between feeling and infant sociomoral evaluation.

Recent research has shown that infants selectively approach prosocial versus antisocial characters, suggesting that foundations of sociomoral development may be present early in life. Despite this, to date, the mental processes involved in infants' prosocial preferences are poorly understood. To explore a possible role of emotions in early social evaluations, the current studies examined whether four samples of infants and toddlers express different emotional reactions after observing prosocial (giving) versus antisocial (taking) events. Experimentally blind coders rated infants' and toddlers' emotional reactions to prosocial and antisocial interactions from video using a 1- to 7-point Likert scale of negative to positive emotion; reactions were rated as more positive after viewing prosocial compared to antisocial interactions in three of four samples. While the observed effects were small, a single-paper meta-analysis suggests that the findings are robust and stable across age. These results support the possibility that emotional reactions play some role in infants' sociomoral evaluations. Statement of contribution What is already known Infants prefer prosocial to antisocial individuals from the first year of life. Emotion plays some role in the sociomoral judgments of children and adults. What this study adds Infants and toddlers express more positive reactions after observing prosocial giving versus antisocial taking acts, though observed effect sizes are small. Naïve coders can predict at a better than chance rate what type of act an infant or toddler just viewed based on their facial expressions. Provides the first evidence that emotion plays some to-be-specified role in infants' and toddlers' sociomoral evaluations.

Social evaluation of intentional, truly accidental, and negligently accidental helpers and harmers by 10-month-old infants.

Whereas adults largely base their evaluations of others' actions on others' intentions, a host of research in developmental psychology suggests that younger children privilege outcome over intention, leading them to condemn accidental harm. To date, this question has been examined only with children capable of language production. In the current studies, we utilized a non-linguistic puppet show paradigm to examine the evaluation of intentional and accidental acts of helping or harming in 10-month-old infants. In Experiment 1 (n=64), infants preferred intentional over accidental helpers but accidental over intentional harmers, suggestive that by this age infants incorporate information about others' intentions into their social evaluations. In Experiment 2 (n=64), infants did not distinguish "negligently" accidental from intentional helpers or harmers, suggestive that infants may find negligent accidents somewhat intentional. In Experiment 3 (n=64), we found that infants preferred truly accidental over negligently accidental harmers, but did not reliably distinguish negligently accidental from truly accidental helpers, consistent with past work with adults and children suggestive that humans are particularly sensitive to negligently accidental harm. Together, these results imply that infants engage in intention-based social evaluation of those who help and harm accidentally, so long as those accidents do not stem from negligence.

Acquiring ownership and the attribution of responsibility.

How is ownership established over non-owned things? We suggest that people may view ownership as a kind of credit given to agents responsible for making possession of a non-owned object possible. On this view, judgments about the establishment of ownership depend on attributions of responsibility. We report three experiments showing that people's judgments about the establishment of ownership are influenced by an agent's intent and control in bringing about an outcome, factors that also affect attributions of responsibility. These findings demonstrate that people do not just consider who was first to possess an object in judging who owns it, and are broadly consistent with the view that ownership is acquired through labor. The findings also suggest that rather than exclusively being the product of social conventions, judgments about the establishment of ownership over non-owned things also depend on the psychological processes underlying the attribution of responsibility.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras.

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers.

In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly(iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN.

Beta common receptor inactivation attenuates myeloproliferative disease in Nf1 mutant mice.

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor, which encodes neurofibromin, a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-, beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly, when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells, concomitant loss of beta c-/- reduced the severity of the MPD, but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML, our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor.

Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules.

Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder.

The NF1 tumor suppressor gene encodes a guanosine triphosphotase (GTPase)-activating protein that negatively regulates Ras signaling and is inactivated in a subset of juvenile myelomonocytic leukemias (JMMLs). Adoptive transfer of fetal liver cells from Nf1 mutant mice models JMML; however, this system has important limitations as a platform for performing biologic and preclinical studies. We have exploited the interferon-inducible Mx1-Cre transgene to ablate a conditional mutant Nf1 allele in hematopoietic cells. Somatic inactivation of Nf1 induces a myeloproliferative disorder with 100% penetrance that is associated with a sub-acute clinical course, tissue infiltration by myeloid cells, hypersensitivity to granulocyte-macrophage colony stimulating factor, hyperproliferation, and resistance to apoptosis. These Mx1-Cre, Nf1(flox/flox) mice establish a tractable experimental model for testing therapeutics and for identifying mutations that cooperate with hyperactive Ras in myeloid leukemogenesis.

Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder.

RAS mutations are common in myeloid malignancies; however, it is not known whether oncogenic RAS can initiate leukemia. We show that expressing mutant K-Ras(G12D) protein from the endogenous murine locus rapidly induces a fatal myeloproliferative disorder with 100% penetrance characterized by tissue infiltration, hypersensitivity to growth factors, and hyperproliferation. Hematopoietic cells from diseased mice demonstrated increased levels of Ras-GTP, but effector kinases were not constitutively phosphorylated and responded normally to growth factors. Oncogenic RAS is sufficient to initiate myeloid leukemogenesis in mice, and this provides an in vivo system for biologic and preclinical studies.

Use of a simple fractionation method to evaluate binding, internalization and intracellular distribution of oligonucleotides in vascular smooth muscle cells.

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4 degrees C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25 kDa polyethylenimine (PEI) as a carrier. After 2.5 h of incubation at 37 degrees C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A large proportion (about 60%) of intracellular radioactivity remained associated with the endocytic compartment. Complexation with PEI completely changed intracellular distributions: the nuclear fraction increased to 70% for PS-ODN. The fractionation method proposed, facilitating study of the subcellular distribution of the ODN, could also be used under appropriate circumstances, to study variations in cytosolic ODN content.

Electroporation-mediated delivery of 3'-protected phosphodiester oligodeoxynucleotides to the skin.

The feasibility of topical delivery in the skin of 3' end modified phosphodiester oligonucleotides using electroporation was investigated. Experiments were performed in vitro, using hairless rat skin. Five pulses of (200 V, 450 ms) were applied. The 3' end modifications of the 15 mer oligonucleotide were: (1) 3'-aminohexyl, (2) biotin, with a triethyleneglycol arm, (3) methylphosphonate links between nucleotides 13, 14 and 15, and (4) 2-O-methyl nucleotides at 13, 14 and 15 positions. All the modifications were efficient to protect the oligonucleotides against degradation in the skin. Electroporation increased the topical delivery of the 3' end-modified phosphodiesters by two orders of magnitude compared to passive diffusion, without significant differences between the derivatives. Oligonucleotide concentrations in the range of 1 microm could be achieved in the viable skin. The delivery of a phosphorothioate congener was lower than phosphodiester delivery due to the interaction of phosphorothioate with the stratum corneum. Consequently, 3' end-protected phosphodiesters could be an interesting alternative to phosphorothioate oligonucleotides for topical treatment of cutaneous diseases.

Parameters controlling topical delivery of oligonucleotides by electroporation.

Electroporation, using high voltage electrical pulses has been recognized as a powerful method for delivering macromolecules such as DNA and proteins in cells, or smaller molecules through the skin. Transdermal electroporation could combine targeted delivery of drugs to the skin and permeabilization of skin cells, suggesting that electroporation could be an interesting alternative for topical delivery of oligonucleotides. This work is devoted to the determination of the electroporation parameters that allow optimal delivery of oligonucleotides to the viable tissues of hairless rat skin in vitro. Phosphorothioate derivatives were preferred to the phosphodiester congeners as the former were found to be much less degraded when extracted from the tissues. Long duration (100-500 ms)--medium voltage (100-200 V)--exponentially decaying pulses appeared to be the best conditions for delivering oligonucleotides to the skin. The oligonucleotide quantity permeating the viable tissues of the skin was controlled by the selection of the electrical parameters of the pulses (voltage, pulse time and number of pulses) or by the ON concentration in the donor compartment. After delivery by electroporation, therapeutic levels of oligonucleotides were reached in the viable tissues of the skin (above 1 microM or 10 microM in intact or stripped skin respectively). Taken together, our results show that electroporation could be an interesting method for the delivery of oligonucleotides to the skin.

Sequence dependency of the internalization and distribution of phosphorothioate oligonucleotides in vascular smooth muscle cells.

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controlled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.

[A simple method for the study of cytosolic content of oligonucleotides in cells]. / Une méthode simple pour étudier le contenu cytosolique en oligonucléotides dans les cellules.

Antisense oligonucleotides are currently used for the specific control of the expression of a selected gene. Their putative targets are located in the cytoplasm (messenger RNA) or the nucleus (pre-messenger RNA or DNA). This approach is conditioned by the presence of the antisense molecule inside the cell at sufficient concentrations and in the appropriate compartments. We propose in this paper a simple method for the study of the cytosolic content of internalized oligonucleotides. This method is based on the selective permeabilization of the plasmic membrane by the detergent digitonin. By complexing to membrane cholesterol, the detergent creates pores through which soluble and diffusible species can escape outside the cells. The selectivity of membrane permeabilization was controlled by using compartment markers: lactate dehydrogenase (LDH) for cytosol, dextrane-rhodamine (DEX) and hexosaminidase (HAM) for endocytic vesicles and lysosomes, respectively. Optimal digitonin concentrations and incubation times have been defined to reach the following pattern of membrane permeabilization: LDH > 80%; DEX and HAM < 15%. The method was applied to monitor the quantity of extractible oligonucleotides from cells after endocytosis. The results showed that phosphodiester and phosphorothioate oligomers are readily available in the cytosol (60-50% of the internalized species), whereas those bearing a hydrophobic moiety (fluorescein, cholesterol) are less diffusible probably owing to membrane binding. Internalization and cytosol partition were found to depend on the chemical nature of the oligonucleotide, and also on the sequence and the cell type. This method could be useful for the selection of antisense molecules that exhibit the best internalization and distribution in cells, and for a more appropriate choice of control sequences in antisense studies.

Targeted photochemical modification of HIV-derived oligoribonucleotides by antisense oligodeoxynucleotides linked to porphyrins.

Antisense oligodeoxynucleotides directed against a 24-mer RNA derived from the long terminal repeat (LTR) region of HIV were linked to proto- and methylpyrroporphyrin and their zinc derivatives. The oligonucleotide-porphyrin conjugates were tested for their ability to induce photodamage on the target RNA. Upon hybridization followed by irradiation at 405 nm, the photochemical reaction led to photocross-linking of the antisense derivative to the RNA substrate. The protoporphyrin exhibited a much higher cross-linking yield than the methylpyrroporphyrin while the Zn-porphyrin derivatives were found to be less efficient than their corresponding nonmetallated congeners. The specificity of the photocross-linking reaction between the porphyrin-oligomer and its target RNA was demonstrated by the following evidence: (1) hybrid formation was required for photocross-linking to occur, (2) the sites of cross-linking on the target RNA were identified at G residues located in close proximity to the porphyrin photoactive center in the hybrid and (3) addition of bulk calf liver RNA did not affect the photocross-linking efficiency.

Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake.

Oligonucleotides can be adsorbed on polyisohexylcyanoacrylate nanoparticles in the presence of hydrophobic quarternary ammonium salts. Oligonucleotides bound to nanoparticles are protected from nuclease attack both in buffer and in cell culture media. Cellular uptake of oligonucleotides is increased when they are adsorbed onto nanoparticles as a result of the capture of nanoparticles by an endocytic phagocytic pathway. Intracellular stability towards nucleolytic degradation is increased in the presence of nanoparticles. These results show that nanoparticles can be considered as convenient carriers for the protection and delivery of oligonucleotides to cells.

Structure-activity relationships for DNA photocleavage by cationic porphyrins.

The influence of molecular structure and DNA binding mode on the ability of cationic porphyrins to photosensitize DNA strand break formation has been studied for a series of meso-substituted pyridinium porphyrins using electrophoretic and DNA sequencing techniques. Porphyrins substituted with pyridyl groups in which the heterocyclic nitrogen is in the para or meta position vis-à-vis the substitution point are capable of intercalative binding and are considerably more efficient DNA photosensitizers than the corresponding non-intercalating ortho compounds. Within each group of porphyrins the photosensitizer efficiency increases with the number of positive charges. Using DNA sequencing experiments, we have demonstrated that photomodification occurs primarily at the guanine and thymine bases, and that alkali-labile sites produced by photo-oxidation are as important as direct cleavage events. The kinetics of strand degradation in aerated and degassed solution suggest that type II reactions (probably mediated by singlet oxygen) occur with significantly higher yield than type I reactions and are responsible for the formation of alkali-labile sites in aerated systems. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization.

Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells.

Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.

Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides.

Adsorption of oligothymidylates on polyisobutyl- or polyisohexylcyanoacrylate nanoparticles was achieved in the presence of hydrophobic cations such as tetraphenylphosphonium chloride or quaternary ammonium salts. Results suggested that oligonucleotide adsorption on the nanoparticles was mediated by the formation of ion pairs between the negatively charged phosphate groups of the nucleic acid chain and the hydrophobic cations. The adsorption efficiency of oligonucleotide-cation complexes on nanoparticles was found to be highly dependent upon several parameters: oligonucleotide chain length, nature of the cyanoacrylic monomer, hydrophobicity of cations used as ion-pairing agents, and ionic concentration of the medium. Carrier capacity of polyisohexylcyanoacrylate nanoparticles for oligothymidylates (16 nucleotides) complexed with cetyltrimetylammonium bromide in the presence of 0.15 M NaCl was determined to be 5 mumol/g polymer. The in vitro protection of oligothymidylates adsorbed to nanoparticles against degradation by a 3'-exonuclease (snake venom phosphodiesterase) was also demonstrated. These results showed that nanoparticles can be considered as convenient carriers for the protection and delivery of oligonucleotides to cells in culture and for future applications in vivo.